One of the most important developments in the aging field has been the realization that many organisms regulate their pace of aging via evolutionarily conserved signaling pathways. These pathways are thought to have evolved early in life's history to delay reproduction under times of stress (e.g. calorie restriction, crowding) and to redirect energy resources to somatic maintenance and defense systems. The budding yeast S. cerevisiae has emerged as a promising model for understanding the molecular basis for the link between calorie intake, stress and longevity. One major cause of yeast aging stems from the instability of the highly repetitive ribosomal DNA (rDNA) locus. Yeast cells suppress this instability by forming "silent" heterochromatin at the rDNA locus. This process requires Sir2, an NAD+-dependent histone deacetylase. Additional copies of the SIR2 gene stabilize the rDNA and extend life span by mimicking calorie restriction. However, in wild-type cells, Sir2 expression is constitutive. This raises the fundamental question: How is Sir2 regulated in vivo? Guarente and colleagues have proposed calorie restriction increases Sir2 activity by decreasing glycolysis which increases total cellular NAD+ levels. However, we have shown that calorically restricted cells (and genetic mimics of calorie restriction) do not have increased steady-state NAD+ levels or altered NAD+/NADH ratios. Moreover, the model cannot explain how many types of stress increase longevity. We have shown that nicotinamide is a potent non-competitive inhibitor of Sir2 in vivo. We propose that during calorie restriction or stress, nicotinamide levels are depleted via a nicotinamidase that is greatly induced under these conditions. Concurrently, a nuclear NAD salvage pathway is also induced, resulting in the activation of an NAD-responsive stress resistance network. The significance of this model is that longevity is an active process rather than simply a response of Sir2 to decreased metabolism. Our aim is to determine how Sir2 is regulated in vivo and identify other transcriptional regulators that are responsive to fluctuations in nuclear NAD+ or its redox state. The focus of my laboratory on the link between NAD+, chromatin structure and silencing places us in a prime position to achieve our goals. As far as we are aware, our approach to understanding this fundamental regulatory network is unique. [unreadable] [unreadable]